Summary Novel Activation Mechanism of the Iys2p of Candida albicans. The alpha-aminoadipic acid pathway for lysine biosynthesis is unique to fungi and not present in bacteria, plants, animals or humans. The pathway consists of eight enzymatic steps and more than ten unlinked genes in Saccharomyces cerevisiae. This pathway is also present in Candida albicans, an important opportunistic fungal pathogen. One of the central enzymes of this pathway, alpha-aminoadipate reductase (AAR), catalyzes the conversion of alpha-aminoadipic acid to alpha-aminoadipic-delta-semialdehyde in the presence of ATP, MgCI, and NADPH. Two distinct gene functions (LYS2 and LYS5) are required for this reaction. The 4.2 kb LYS2 gene encodes 150 kDa AAR and contains all of the functional domains required for AAR catalytic activity. The function of the 0.9 kb LYS5 gene was unknown until recently. Sequence analysis of the C. albicans LYS2 gene revealed a highly conserved novel posttranslational activation domain (LGGHSI, amino acids 880 to 885) for the phosphopantetheinylation of serine residue 884 in the presence of CoA by an unique phosphopantetheinyltransferase (PPTase) encoded by the LYS5 gene. This type of posttranslational activation is highly unusual (no such example exists) for any amino acid biosynthesis enzyme. The importance of the activation domain in the Lys2p and the PPTase domain (G-D---W--KES--K) in the Lys5p has not been analyzed by site-directed mutagenesis. In this project, I propose to isolate and express the cloned C. albicans LYS2 and LYS5 genes in E.coli BL-21, determine the molecular mass of Lys2p and Lys5p, demonstrate posttranslational activation of the inactive Lys2p (AAR) in the presence of CoA and PPTase in vitro and in vivo, and perform site-directed mutational analysis of the specific amino acid residues in the activation domain of Lys2p and PPTase domain of Lys5p. This investigation is essential for understanding unusual posttranslational activation mechanism of the LYS2 encoded AAR. This study will also serve as a model for investigation of this unique pathway in other pathogenic fungi and provide basic knowledge on unique genes and proteins as potential targets in the design of molecular probes for fungal detection and development of antifungal drugs in the future.